Image forming cytometer

ABSTRACT

The present invention relates to methods and systems for image cytometry analysis, typically at low optical magnification, where analysis is based on detection of biological particles using UV bright field and optionally one or more sources of excitation light.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a Continuation of U.S. Ser. No. 14/893,780, filed onNov. 24, 2015, which is a U.S. National phase application ofInternational application no. PCT/DK2014/050151, filed May 28, 2014,which claims priority to DK PA 2013 70291, filed on May 28, 2013; thecontents of each of which are incorporated herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods and systems for image cytometryanalysis, typically at low optical magnification, where analysis isbased on detection of biological particles.

BACKGROUND OF THE INVENTION

Microscopy has been used for the analysis of biological material for along time. In order to see an object in a microscope it is necessarythat the object displays optical properties that differs from theoptical properties of the background and this difference is calledcontrast. Biological particles are typically largely made up of water,contained within the cell membrane, which makes them inherently similarto their surroundings. The interior of the cell differs typically fromthe surrounding liquid by certain chemical constituents, such asproteins, DNA and RNA, some of which form “structure” which is of suchsize that it can potentially be visualised, for instance DNA packed intoa cell nuclei. Biological particles, such as mammalian cells, yeast andbacteria are relatively small, typically less than about 20 μm indiameter, which can make them difficult to view in microscopy unlesssome advanced techniques are applied. Among such techniques are highmagnification, phase contrast and UV microscopy and with theintroduction of digital technology several image enhancement techniqueshave been introduced.

High magnification microscopy typically uses magnification of ×50 ormore, which makes it possible to separate the minute structures whichaids the visualisation and identification of the biological particles.Phase contrast microscopy exploits small differences in refractive indexto produce an image with high contrast. UV microscopy uses absorbanceproperties of proteins and DNA, which absorb light at around 260 and 280nm respectively. The absorbance of light is seen as contrast in themicroscope. Further light of short wavelength makes it possible toseparate smaller structures than is possible using light of longerwavelength since the maximum resolution of the microscope is dependenton the wavelength of the light. Such small structures in biologicalparticles are typically the internal structures of a cell, such as anucleus.

In assessment of biological particles by Image Cytometry it is ofparamount importance to know the location of biological particles in theimage. This is of course obvious when the task of analysing biologicalparticles is the enumeration of particles in a sample but this is alsothe case in most any assessments concerning other properties of samplesand/or cells. A necessary condition for the identification of any objectin an image is that it is possible to establish conditions where thereis a significant difference in the image of the object and that of thesurrounding background.

Typical methods of microscopy are based on optical properties which donot modify the wavelength of light, such as difference in refractiveindex, reflectivity or attenuation, while methods such as fluorescencemicroscopy are based on shift in wavelength of light, typically broughtabout by quantum mechanical properties of matter.

In microscopy such difference is generally referred to as “contrast”.There are several methods to produce contrast in microscopy, the twobasic methods being Dark Field (DF) and Bright Field (BF) microscopy,where the intensity of the “Field” signal refers to the intensity of thebackground, that is the region of the image separating any objects whichmight be present. Therefore in DF the background is dark and the objectshave higher intensity, contrary to BF where the background is luminousand the image of objects represents decrease in light.

When considering microscopy analysis of biological particles, such asbiological cells both DF and BF microscopy methods render images ofrather poor contrast. Therefore there are additional techniques whichare widely used in the analysis of biological particles since theygenerally offer greater contrast in the images, such as phase contrastand fluorescence microscopy. Both methods have advantages as well asdrawbacks when it comes to implementation for the identification ofbiological particles. Phase contrast microscopy requires specialisedoptical components, while fluorescence microscopy is limited byselectivity defined by the fluorophore system used, which either must bepresent in the particles or bound to the particles.

SUMMARY OF THE INVENTION

Present invention offers a simple, effective and reliable method torecord images of biological particles with considerable contrast, whichmakes methods and system according to the invention particularly wellsuited for the identification of particles in Image Cytometry.

The present invention provides an image cytometer, comprising:

-   -   a first light source configured for emitting light into a sample        region;    -   focusing means for forming collimated light and directing the        collimated light from the first light source along an optical        axis of the cytometer;    -   a second light source comprising a first excitation light source        configured for emitting excitation light into the sample region;        and    -   image forming means for forming an image of at least part of the        sample region on an array of detection elements, wherein    -   the sample region is located between the focusing means and the        array of detection elements, and wherein    -   the cytometer is able to be configured to be interchanged        between a bright field mode, a dark field mode and a        fluorescence mode, and/or wherein    -   the first light source is configured for emitting light with a        wavelength less than 400 nm, and/or wherein    -   the excitation light is at an incidence angle relative to the        optical axis so as to provide the fluorescence mode.

The present invention provides further an illumination system for animage cytometer, comprising:

-   -   a first light source configured for emitting light;    -   focusing means for directing the light from the first light        source along an optical axis of the image cytometer and into a        sample region; and    -   a second light source comprising a first excitation light source        configured for emitting excitation light and into the sample        region, wherein    -   the light from the first light source is configured for emitting        light with a wavelength less than 400 nm.

Even further, the present invention provides a method for the assessmentof at least one quantity parameter and/or one quality parameter of abiological sample, comprising:

-   -   applying a volume of the biological sample to a sample        compartment having parallel wall parts defining an exposing        area, the wall parts allowing light from an image cytometer to        pass through the wall parts of the sample compartment,    -   illuminating the sample compartment with light from the first        light source, and exposing, onto a 2-dimensional array of active        detection elements, light having passed through the sample        compartment, thus recording an image of spatial light intensity        information,    -   illuminating the sample compartment with excitation light from        the second light source, and exposing, onto the 2-dimensional        array of active detection elements, fluorescent light having        passed through the sample compartment, thus recording a        fluorescent image of spatial light intensity information,    -   processing both images in such a manner that light intensity        information from individual biological particles are identified        as distinct from light intensity information from the        background,    -   and correlating the results of the processing to the at least        one quantity parameter and/or quality parameter of biological        particles in the biological sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an embodiment of an image cytometer according to thepresent invention.

FIG. 2A shows a graph that illustrates observed contrast.

FIGS. 2B, 2C, 2D, 2E, 2F, and 2G illustrate Bright-field images recordedusing different wavelength of light.

FIGS. 3A, 3B, 3C, 3D, 3E and 3F illustrate arrangements of inclinedlight sources and effect of masking of the light.

FIG. 4 shows a graph that illustrates intensity of fluorescent polymerbeads.

FIGS. 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, 5I, 5J, and 5K illustrateconfiguration of passive light modulation in Bright-field images.

DETAILED DESCRIPTION OF THE DRAWINGS

One first effect of the image cytometer according to one embodiment isthat the image cytometer is able to be configured to be interchangedbetween a bright field mode, a dark field mode and a fluorescence mode.In a preferred embodiment of the present invention, the first lightsource may provide the bright field light source and the dark fieldlight source, such that only a single light source is required for thebright field mode and the dark field mode. The interchanging between thetwo modes may thus be obtained by interchanging means rather thanchanging of light source or optical means. Preferably the first lightsource is configured for emitting light with a wavelength less than 400nm. In this perspective, the first light source may be regarded as anultraviolet (UV) bright field light source. It has been found that aneffect of using a UV bright field light source is that such illuminationprovides more details about the particles, and/or higher contrast in animage, in comparison to images recorded with light from a light sourceemitting light with a higher wavelength than 400nm.

According to the present invention, the second light source may providethe fluorescence mode, and an effect of the present invention is thatthe fluorescence mode may be provided without moving the second lightsource or parts that may direct fluorescence light from the second lightsource. In other words, the fluorescence mode may be provided rapidlysince there may be no movement of a fluorescence light source or nomovement of parts that may direct fluorescence light. Preferably, theexcitation light is at an incidence angle relative to the optical axis,the optical axis of the image cytometer being defined by the axisbetween the sample region and the array of detection elements, so as toprovide the fluorescence mode. It has been found that one effect of sucha setup is that the intensity of exposed excitation light onto thedetection elements may be reduced significantly.

Attenuation Means and Modulation Means

In preferred embodiments of the present invention, the image cytometerfurther comprises attenuation means, such as an optical filter such asto attenuate the light intensity in one or more predefined wavelengthband(s), preferably where attenuation means are placed at apredetermined plane along the optical axis. Even further, the imagecytometer may further comprise modulation means, such as a spatialmodulation means, preferably where modulation means are placed at apredetermined plane along the optical axis. In some embodiments of thepresent invention, the attenuation means and/or the modulation meansis/are placed in the light path between the first light source and thesample region. Preferably, the attenuation means and/or the modulationmeans is/are placed in the light path between the sample region and thearray of detection elements. More preferably, the modulation means areplaced in or close to the focal plane of collimated light transmittedthrough the sample region towards the detection elements along theoptical axis.

It has been found that there are many effects of having modulation meansplaced in or close to the focal plane of the collimated lighttransmitted through the sample region are many. First of all, thecontrast, such as at high spatial frequencies, may be improved comparedto an arrangement without modulation means at this position. Secondly,since the focal plane of the collimated light may be the aperture stopof the image-forming device, the modulation means may yield extendeddepth of field. Thirdly, the modulation means may allow only the lightrefracted or dispersed by particles in a sample in the sample region toreach the detection elements and eliminating light emitted directly fromthe light source. By an arrangement where the modulation means is placedin or close to the focal plane of the collimated light transmittedthrough the sample region it may be possible to realise an imagecytometer that has the flexibility of recording high contrast brightfield or dark field images, or the combination of the two, simply byplacing or removing a suitable modulation.

In some embodiments of the present invention, two or more of theattenuation means and/or the modulation means is/are mounted ininterchanging means that allow removal or interchanging of theattenuation means and/or modulation means. The interchanging means mayfor example comprise a rotating unit, such as a filter wheel. The two ormore interchanging means may be such that none, one, two or moreattenuation and/or modulation means can be positioned along the opticalaxis at the same time. An effect of having two or more interchangingmeans may be that these allow for combined effects of modulations means,attenuations means or both means. Another effect may be that two or moreinterchanging means may interchange the attenuation means and/or themodulation means more rapidly than a single interchanging means havingthe same number of attenuation means and/or modulation means, forexample since such interchanging means may be smaller, and able torotate faster than the single interchanging means.

In some embodiments of the present invention, at least one modulationmeans may be partly opaque or partly transparent. The attenuation and/orthe modulation means may likewise be partly opaque in some part(s) andpartly transparent in different part(s), preferably where one or more ofthe parts is/are circular in shape. Accordingly, part of at least onemodulation means may be partly opaque and another part of the modulationmeans may be partly transparent. Preferably, the attenuation means mayattenuate light by a predetermined factor, such as less than 10⁻³, suchas less than 10⁻⁴, such as less than 10⁻⁵, or such as less than 10⁻⁶.

In a preferred embodiment of the present invention, the modulation meanscomprises an obstruction configured for the dark field mode, preferablysubstantially attenuating collimated light passing through the sampleregion. In another preferred embodiment of the present invention, themodulation means comprises an aperture configured for the bright fieldmode, preferably substantially attenuating uncollimated light emittingfrom the sample region. It may be achieved by interchanging of theobstruction and the aperture, that the interchanging between the brightfield mode and the dark field mode is possible. Interchanging between abright-field mode and a dark-field mode may thus be realised bymodulation means, preferably by inserting and/or interchangingmodulation means located between the sample region and the array ofdetection elements.

In another preferred embodiment of the present invention, the modulationmeans comprise phase contrast microscopy modulation means. Accordingly,the modulation means may constitute phase contrast microscopy modulationmeans and the substantial wavelength of the light emitted by the firstlight source is of a narrow waveband, preferably where the width of thewaveband is less than 50 nm.

First and Second Light Sources

In optical setup, it may be accepted that a perfectly collimated is notalways possible to obtain, and the collimated light from the first lightsource may deviate from collimated light with a deviation angle lessthan 10 degrees, more preferably less than 5 degrees.

In embodiments of the present invention, the wavelength from the firstlight source is between 200 nm and 700 nm. Preferably, the wavelengthfrom the first light source may be between 300 nm and 395 nm. Even morepreferably, the wavelength from the first light source may be between320 nm and 380 nm. Most preferably, the wavelength from the first lightsource may be between 350 nm and 380 nm.

The excitation light from the secondl light source may have a wavelengthsubstantially different from the wavelength of light from the firstlight source. Preferably, the incidence angle of the excitation lightmay be between 10 and 80 degrees, preferably between 20 and 60 degrees,and more preferably between 30 and 50 degrees. Alternatively, theincidence angle of the excitation light may be 90 degrees. In anotheralternative embodiment of the present invention, the incidence angle isbetween 110 and 180 degrees, preferably between 120 and 160 degrees, andmore preferably between 130 and 150 degrees.

Focusing Means

In a preferred embodiment of the present invention, the focusing meanscomprises a lens, whereas in another equally preferred embodiment of thepresent invention, the focusing means comprises a curved mirror.

Additional Light Sources

In some embodiments of the present invention, the image cytometerfurther comprises an additional light source, such as a third or fourthor fifth or sixth light source, preferably where the additional lightsources are excitation light sources. The light source(s) may be a lightemitting diode and/or a diode laser and/or a laser such as tuneablesolid-state light source(s) and/or a tuneable light emitting diode. Thetuneable solid-state light source may be a tuneable laser diode.

Optical Means and Detection Elements

In preferred embodiments of the present invention, the light source(s)is/are optically connected to optical means configured for providinglight with a substantially uniform intensity across the sample regionand/or across a region imaged by the array of detection elements. Theoptical means may comprise an array of micro lenses. Alternatively, Theoptical means may comprise an array of cylindrical micro lenses,preferably it may comprise two arrays of cylindrical micro lenses withsubstantially perpendicular orientation of the cylindrical lenses. Thearray of detection elements may be an array of CCD or CMOS sensorelements.

Exposures

According to the present invention, the light source(s) is/areconfigured for emitting light in duration less than 1 second, preferablyfor less than 0.1 second. Preferably, the light source(s) is/areconfigured for emitting light in duration between 0.0001 and 0.1000second, preferably between than 0.0001 and 0.0500 second. However, insome situations such as when high sensitivity is required influorescence imaging, the light source(s) may be configured for emittinglight in duration for more than a 1 second, such as for more than 2seconds, such as for more than 3 seconds, such as for more than 4seconds, such as for more than 5 seconds, such as for more than 6seconds, such as for more than 7 seconds, such as for more than 8seconds, such as for more than 9 seconds or such as for more than 10seconds.

Light Blocking

In one embodiment of the present invention, light from the excitationlight source may substantially be eliminated from reaching the entranceof the image forming means by selectively removing rays of light fromthe beam of excitation light. The rays may be selectively removed byplacing one or multiple obstructions in the beam of excitation light,preferably where the beam of excitation light is substantiallycollimated in the plane where the obstruction is placed.

Image Forming Means

In a preferred embodiment of the present invention, the image formingmeans is configured for providing a depth of field that is more than 5μm, such as between 10 μm and 150 μm. In this way, the sample region maybe in focus in the depth of field such that the image forming meansand/or the array of detection elements may not need to be moved in orderto acquire a sharp image of a sample in a sample region, for examplewhen the sample has particles positioned at different depths. However,in some embodiments of the present invention, the image forming meansand/or the array of detection elements and/or the sample compartment maybe configured for moving such that image forming means and/or the arrayof detection elements may be placed at an optimal position relative thesample. One sample or a part of a sample may for example be in focus inone configuration, but when changing to another sample or to a differentpart of a sample, the other sample or the different part of the samplemay then not be in focus, and it may thus be required to either move thesample and/or the image forming means and/or the array of detectionelements.

In preferred embodiments of the present invention, the image formingmeans is configured for transmitting light in the wavelength region ofbetween 200 nm and 1000 nm, more preferably in the wavelength region ofbetween 350 nm and 1000 nm, more preferably in the wavelength region ofbetween 350 nm and 850 nm.

In several preferred embodiments of the present invention, the imageforming means comprises a microscope objective. The image forming meansmay be configured for providing a linear enlargement of the sample.Preferably, the linear enlargement is smaller than 20:1. The linearenlargement may also be in the range from 1:1 to 20:1, preferably in therange from 1:1 to 10:1, more preferably in the range from 1:1 to 4:1.

In several preferred embodiments of the present invention the imagecytometer is configured with means which allow two or more image formingmeans to be interchanged between recording of images. The purpose ofinterchanging imaging means is preferably to change the opticalproperties of the image cytometer, such as to change linearmagnification of the image and/or to change the depth of field of view.Often the selection of imaging means is made on the basis of a prioriknow properties of a sample, but in several preferred embodiments of thepresent invention the selection of imaging means is made on the basis onresults of an assessment of the sample being analysed.

Sample

The sample region may comprise a sample such as a biological sample. Thesample may be in a sample compartment.

Illumination System

According to the present invention, the illumination system may be forproviding the required light sources to an image cytometer, being abright field light source, and a fluorescence light source. In apreferred embodiment of the present invention, the focusing means inrelation to the illumination system is for forming collimated light fromthe first light source. Furthermore, the excitation light may be at anincidence angle relative to the optical axis. The illumination systemmay additionally have any of the features from the image cytometer aspreviously described.

Cytometry Method

In one aspect of the present invention, there is provided a method forthe assessment of at least one quantity parameter and/or at least onequality parameter of biological sample, comprising

-   -   applying a volume of the sample to a sample compartment having        parallel wall part defining an exposing area, the wall part        allowing light from a first light source to pass through the        wall parts of the sample compartment,    -   exposing, onto an array of active detection elements, light        having passed through the sample compartment, thus recording an        image of spatial light intensity information,    -   processing the image in such a manner that light intensity        information from individual biological particles are identified        as distinct from light intensity information from the        background,    -   and correlating the results of the processing to the at least        one quantity parameter and/or the at least one quality parameter        of biological particles in a liquid sample.

The present invention relates to methods and systems for the assessmentof quality and/or quantity parameter of biological samples, includingoptical interaction with the sample. The optical interaction with thesample preferably causes alteration in intensity and/or direction oflight as a result of interaction with a biological sample, or particlesin said biological sample, some of the preferred interactions being oneor more of the following; reflection, refraction, diffraction,interaction, scattering or absorbance.

In preferred embodiments of the present invention the biological samplebeing assessed is contained in a sample compartment. A preferredproperty of the sample compartment is to define a boundary of thesample. Another preferred embodiment of the present invention is wherethe wall part of the sample compartment is the bottom of an opencontainer. In several embodiments the sample compartment the boundary isformed by a transparent wall part defining either bottom or top of thesample, while in other equally preferred embodiments the samplecompartment is formed by two transparent wall parts, where the sample isplaced in between the wall parts, defining the thickness of the samplebeing assessed.

In one preferred embodiment of the present invention the biologicalsample being assessed is a suspension of biological particles. Suchsuspension of biological particles can be a portion of a larger samplevolume where the purpose of the assessment can be to determine orestimate the property of the larger sample volume. In another equallypreferred embodiment, the biological sample is a sample of cells grownand/or growing on a substrate. Alternatively, the sample may be a liquidsample. In some preferred embodiment the substrate is in suspension,while in other equally preferred embodiment such substrate is, or canbecome, an integrated part of the sample compartment. Generally it ispreferred that substrate placed in the sample compartment issubstantially transparent and in many preferred embodiments thetransparent substrate plate is a wall part of the sample compartment.

In preferred embodiment of the present invention the assessment of atleast one quantity parameter and/or at least one quality parameter ofbiological sample is the analysis of individual cells. Such individualcells are often isolated, either in suspension or on a substrate, butsuch individual cells can also be in a clump of cells, such cellsadhering to each other. In other preferred embodiments of the presentinvention the assessment of at least one quantity parameter and/or atleast one quality parameter of biological sample is the analysis of bulkof cells, such as a tissue sample.

In several most preferred embodiments of the present invention the firstlight source is a light emitting diode (LED) and/or a diode laser and/orlaser. Several of the properties of LED's and laser diodes offersubstantial advantages in the design and operation of system accordingto the present invention, such as small physical size and powerefficiency. In many preferred embodiments of the present invention thewavelength of the light from the first light source is less than 400 nm.It is often preferred that the wavelength of the light from the firstlight source is between 200 nm and 400 nm, such as between 300 nm and395 nm. It has surprisingly been found that the use of light of shortwavelength offers substantial improvement in the assessment ofbiological particles according to the present invention and light inwavelength bands such as 200 nm to 250 nm, 200 nm to 300 nm, 250 nm to350 nm, and 320 nm and 380 nm are all preferred.

Often the biological particles being assessed are sensitive to light, toa degree where it can alter the properties of the particles, and onepreferred method to reduce the effect of the light is to limit thelength of time the sample is exposed to the light is to limit the timethat the light source emits light onto the sample, preferably where theduration of illumination of light from the light source is less than 1second, more preferably where it is less than 0.1 second. In otherequally preferred embodiments illumination period is between 0.0001 and0.1000 seconds, such as between 0.0001 and 0.0500 seconds. Expressed inenergy, it is preferred that the sample is illuminated with 200 nJ/mm²or less, such as 100 nJ/mm² or less, preferably 50 nJ/mm² or less, suchas 20 nJ/mm² or less during exposure.

One preferred method for the assessment of biological particles,according to the present invention, is based on recording an image ofspatial light intensity information from the volume of sample wheresignal from individual biological particles is attenuated lightintensity signal relative to light intensity from the background. Theattenuation can be brought about by one or several of the following,reflection, refraction, diffraction, interference, absorption,scattering. In these embodiments the light relating to a biologicalparticle is lower in intensity than the signal from the background andon the bases of this it is possible to process the image in a mannerwhere the signal from individual cells and signal from the backgroundare distinct from each other, preferably where the signal from theparticle is substantially less than the signal from the background,preferable signal from the background in close spatial proximity to theparticle.

In several preferred embodiments the attenuation of light is caused bythe scattering of light, such scattering originating in processes suchas refraction and/or reflection of light. Further in these and otherpreferred embodiments attenuation is caused by the absorption of light,such absorption being caused by chemical constituents of the biologicalparticles under assessment and/or from other chemical constituents whichare intentionally added to the sample. Accordingly, absorption may becaused by a reagent added to the sample. In embodiments according to thepresent invention attenuation of between 5% and 70% of the lightassociated to biological particles, relative to the intensity of signalsfrom the background, is realised. In other equally preferred embodimentsthe attenuation of light associated to biological particles relative tolight from the background is of between 50% and 90%.

In other equally preferred embodiments of the present invention signalfrom individual biological particles is enhancement, e.g. observedincrease in light intensity signal relative to light intensity from thebackground. This can be caused by processes such as scatter,interference, reflection and refraction, typically in combination withfocusing or other alteration of the light signal, the enhancement beingthe result of spatial re-distribution of light. In these embodiments thelight relating to a biological particle is higher in intensity than thesignal from the background and on the bases of this it is possible toprocess the image in a manner where the signal from individual cells andsignal from the background are distinct from each other, preferablywhere the signal from the particle is substantially higher than thesignal from the background, preferable signal from the background inclose spatial proximity to the particle.

In yet other highly preferred embodiments of the present invention therecorded image of light intensity from biological particle comprisessignals relating to biological particles, such images comprises changein light intensity information which is a combination of attenuation andenhancement of light intensity relative to the light intensity from thebackground.

An often preferred method of the present invention, which generally hasthe effect of increasing the contrast in the recording of lightintensity information, is to modulate light transmitted or scatteredthrough the sample. Preferably such modulation corresponds to spatialdifference in light property at a predetermined plane in the light-pathfrom the light source to the array of active detection elements. Suchmodulation is typically brought about through the use of modulationmeans, such as means that are opaque or substantially opaque, ortransparent, preferably where the degree of transparency, e.g.attenuation is a predetermined property. Preferably such modulationmeans are implemented as a combination of opaque and transparent meansthus the modulation means being opaque at predetermined locations, orregions, while it is transparent at other predetermined locations, orregions. Two preferred implementations of modulation means are firstlyan opaque disk with a hole in its centre and secondly an opaque diskwith diameter which is substantially less than the diameter of theparallel beam of light. When these two modulations means are used incombination then the dimension of the centre hole in the first means aresimilar or identical to the dimension of the disk in the second means.Preferably the predetermined location of opaque and/or transparentregions corresponds to an image of the light source at an optical planein the vicinity of the location of the modulation means. Preferably suchmodulation means have different effect on light transmitted through thesample, and light being transmitted through a biological particle.Typical preferred location of modulation means are close to a focalplane of parallel light entering the collection objective of the imagingsystem.

Typically preferred properties of modulation means are those which alterthe light passing through the modulation means, such as; change inphase, change in intensity, e.g. attenuation, masking of light, e.g.blocking of light. Modulation means preferably include one or several ofthese properties.

In may preferred embodiments modulation means are placed along theoptical axis at location between the light source and the sample, whileit is equally preferred to place modulation means at location along theoptical axis between the sample and the array of active detectionelements. Still may preferred embodiments include modulation means onboth sides of the sample, along the optical axis. In embodimentsincluded modulation means only at location along the optical axisbetween the sample and the array of active detection elements, the lightfrom the light source is substantially parallel as it traverses thesample, preferably also the light intensity of such parallel light issubstantially even across the portion of the sample being imaged by thearray of active detection elements.

When optical components such as lenses are used to focus light from thesample compartment onto the array of active detection elements, parallellight transmitted through the sample compartment is often focused at aplane along the optical axis. Under these conditions it is oftenpreferred to place modulation means at, or close to, such focus plane.In preferred embodiments light which has interacted with a biologicalparticle, causing it for instance to be deflected, refracted and/orscattered, enters the collection objective of the imaging system.Further it is often preferred that the properties of the modulationmeans, such as opaque and transparent properties, are arranged tosubstantially follow the shape of the image of the light source at thisfocus plane. Often preferred properties and form of the modulation meansis a difference in net effect of light transmitted through the samplecompartment and light transmitted through a biological sample.

In embodiments including modulation means it is generally preferred touse light source that produce light consisting of a predeterminedwaveband of light, preferably where the waveband is substantiallynarrow, such as no more than 50 nm, preferably even less than 30 nm inwidth, such less than 20 nm. Further it is typically preferred to uselight at predetermined wavelength, preferably where the wavelength ofthe light is predominantly less than 400 nm, such as between 250 nm and400 nm, more preferably between 300 nm and 390 nm.

Preferred embodiments of the present invention include modulation meansarranged in a manner which produces a phase contrast image, but underconditions where the wavelength of the light transmitted through thesample compartment is less than 400 nm.

Several preferred embodiments of the present invention includemodulation mean that can be placed or removed from the light path, oftenpreferably where two or more different modulation means are included.Accordingly, the two or more images of spatial light intensity may berecorded where two or more substantial different modulation means areapplied. In such embodiments, where it is possible to alter the lightpath by including or omitting modulation means it is preferred to recordtwo or more images of light intensity information from light transmittedthrough the sample compartment. Accordingly, the two or more images ofspatial light intensity information, recorded using the first lightsource, may be used in the processing of light intensity information.

In a preferred embodiment light from a light source passed through thewall part of the sample compartment has general orientation that issubstantially perpendicular to the surface of the wall part. In anotherpreferred embodiment of the present invention, the light from the lightsource is substantially parallel as it passes the sample. In yet anotherpreferred embodiment of the present invention, the collimated lighthaving passed the sample is substantially focused to a plane locatedbetween focusing means and array of detection elements where modulationmeans are placed substantially in the focus plane. As light from a lightsource is preferably guided by the use of one or more opticalcomponent(s) such as a lens(es) and/or mirror(s), this orientation canbe regarded as the optical axis of the light source and in that contextthe optical axis of the light source is substantially perpendicular tothe surface of wall part. Further in several embodiments it is oftenpreferred that the wall parts of the sample compartment is substantiallyperpendicular to the direction of view of the array of active detectionelements. Similarly, as light is generally focused onto the array ofactive detection elements through the use of optical components such aslens(es) and/or mirror(s), this corresponds to that the optical axis ofthe array of detection elements is perpendicular to the wall parts. Thusseveral preferred embodiments of the present invention comprise a lightsource, sample compartment and array of active detection elements thatare all substantially located on a single axis, such that the opticalaxis of a light source and the array of detection elements aresubstantially on a single axis, this axis passing through the samplecompartment such that its wall parts are substantially perpendicular tothis common axis.

Many highly preferred embodiments of the present invention include twoor more light sources for the illumination of the sample. The two ormore light sources typically differ in properties, such as arrangementof optical axis or wavelength. One preferred embodiment of the inventionincluding two or more light sources is where a light from a second lightsource is passed through wall part of the sample compartment, where thesecond light source is arranged in a manner such that the main directionof light enters through wall part defining an exposing area of thesample compartment at an angle, preferably at an angle of between 10 and80 degrees relative to perpendicular. Such arrangement offers typicallyadvantage when two or more light sources are mounted at a substantiallypermanent location, in particular when more than two light sources areto be mounted. Further it has been found that such angular arrangementcan reduce intensity of background light signal. Further, severalpreferred embodiments of the present invention include three or more,such as four light sources illuminating and/or passing light through thewall parts of the sample compartment. Other equally preferredembodiments include more than four light sources, such as five lightsources. As the increasing number of light sources offer the possibilityof performing an assessment of biological particles, where theprocessing is based on plurality of light intensity information,embodiments including six, seven, eight or even more than eight such asten individual light sources are preferred.

Here, and elsewhere in the present discussion, the term “main directionof light” can be interchanged with the term “optical axis”, whichtypically is formed by arranging a light source, or another activecomponent such as an array of active detection elements, in combinationwith optical means such as one or more and/or mirror(s), (es),irrespective of the function of the lens(es) and/or mirror(s) whether itbe for collimation, focusing or dispersion. The optical axis of such asystem generally has an axis of symmetry.

In preferred embodiments of the present invention two or more individuallight sources emit substantially identical light, for instance withrespect to wavelength, preferably in situation such as where such two ormore light sources are operated in substantial synchronisation and thusincreasing the total light energy emitted, and/or extending theilluminated area of the sample compartment, and/or assuring morehomogeneous light intensity illumination of at least a part of thesample compartment. In these but also in other equally preferredembodiments of the present invention two or more individual lightsources emit substantially different light, for instance with respect towavelength, preferably in situation such as where such two or more lightsources are operated independently, such as only one such light sourcebeing turned on at a time, or such that only the light of one such lightsource is illuminating the sample compartment at a time. One preferredproperty of such two or more light sources emitting different light isthat at least one of such light sources give rise to fluorescence,preferably where such fluorescence intensity information is used incombination with attenuated light intensity information for the purposeof assessing at least one quantity parameter and/or at least one qualityparameter of biological sample.

In several preferred embodiments, all the light sources are located atthe same side of the sample compartment. In such embodiments it is oftenpreferred that such light sources are located at the opposite side ofthe sample compartment from the array of active detection elements.

Often it is preferred that two or more light sources are arranged insuch a manner that light sources giving rise to fluorescence are placedat an angle to the wall parts of the sample compartment, such that theoptical axis of the light source are substantially not perpendicular tothe wall parts of the sample compartment. One preferred advantage ofsuch an arrangement is the increase in the ratio of the intensity offluorescence light emitted from biological particles to the intensity oflight from the background, often termed Signal to Background (S/B).Light from the background, where background typically is/are region(s)in the image of light intensity information outside any biologicalparticle of interest, can originate from of a number of sources and/orphenomena some of which are directly related to the properties of thelight source, such as the orientation of the light source relative tothe optical axis of the array of detection elements. Another equallypreferred advantage of arranging a light source at an angle to the wallparts of the sample compartment is that it is possible to locate anumber of light sources at a fixed position relative to the samplecompartment, thus allowing illumination of the sample compartment withlight from two or more light sources without use of mechanical means.Preferred advantage of such properties is/are more simple constructionof a system and/or the ability to operate the system faster, when thetask is to illuminate the sample in the sample compartment with two ormore different wavelengths in sequence. Further it has been found thatsuch arrangement of excitation light source reduces internal reflectionfrom the light emitting onto a plan surface of the light source, whichotherwise can be reflected back onto the array of active detectionelements.

One preferred embodiment of the present invention is the use of a lightsource for the recording of attenuation of light, for instance throughrefraction and/or reflection. Often it is preferred that the light fromsuch a light source is transmitted through the sample compartment in asubstantially collimated manner, such that a substantial portion of thelight is parallel or substantially parallel. It has been surprisinglyfound that such substantially parallel light can enhance the contrast ofthe attenuation, that is the ratio of attenuation of light by thebiological particle to the intensity of transmitted light in a region ofthe background. In these and other preferred embodiments of the presentinvention a portion of the light transmitted through the sample is notexactly parallel, but preferably at an angle less than 45° relative toparallel, such as less than 30°, such as 15°. Even less divergence, suchas 10° or less, is often preferred, such as divergence of light of nomore than 5° relative to the optical axis.

It has been found that in several preferred embodiments the highcontrast recorded under collimated conditions can be substantiallymaintained while a moderate divergence often contributes positively toevening out the intensity of light transmitted through the samplecompartment and exposed onto the array of active detection elements.Often many of these embodiments have the focus point of the lightsource, brought about by an optical components such as lens(es) ormirror(s), substantially outside the sample compartment.

In other equally preferred embodiments of the present invention, for therecording of attenuation of light, the light from a light source passingthrough the sample compartment for the recording of attenuation of lightis substantially focused on the sample compartment. Preferably where asubstantial portion of the light transmitted through the samplecompartment can be recorded by the array of active detection elements.

One feature of the present invention, which is highly preferred is thatthe light transmitted through, and/or onto, the sample compartment issubstantially even in intensity across the field of view of the array ofactive detection elements. Preferably deviation from even illumination,for instance expressed as the ratio of the variation of intensity to themean intensity, is less than 25%, more preferably less than 10% and evenmore preferably less than 5%. Such property generally has the effect ofreducing variation in an optical property of a sample as recorded on thearray of active detection elements, which often is results in lowervariation in the expression of a property, which substantially isdependent on the intensity of illuminated light. This is for instanceapparent for both attenuation of light and emission of fluorescence.

Optical means, consisting of number of lenses arranged in an array, canbe arranged to substantially focusing multiple images of thelight-emitting element of a light source onto and/or through the samplecompartment. Such arrays of micro lenses are preferred in severalembodiments of the present invention for the purpose of effectivelyilluminating a portion of the sample compartment, preferablysubstantially only illuminating a portion from which light is exposedonto an array of active detection elements.

One often preferred arrangement of micro lenses includes an array ofcylindrical micro lenses. Such a micro lens array if preferably used incombination with a second or more array(s) of cylindrical micro lenses,which typically are oriented perpendicular to each other. Such anarrangement is included in several preferred embodiments of the presentinvention, mainly where it improves the efficiency of illumination bycreating an even illumination across a part of the sample compartmentand/or by transmitted a high fraction of the light emitted from thelight source onto a part of the sample compartment. In several preferredembodiments the properties of such two or more arrays of cylindricalmicro lenses are substantially identical, while in other often-preferredembodiments the properties are substantially different, such as toproduce illumination which is adapted in shape to the shape of the arrayof active detection elements. Properties of arrays of micro lenses thatare varied to produce such shape are for instance pitch of the lensesand/or the focal length.

It is often preferred to include optical means to focus light exposedonto an array of detection elements, such as one or more lens(es) ormirror(s), where such optical means have an ability to focus exposedlight signals, expressed as depth of focus in the object plane. Inseveral preferred embodiments of the present invention such focusingmean have focus depth larger than 5 μm, preferably in the range from 10μm to 150 μm.

In order to use a single optical means for both the recording ofattenuated transmitted light and emitted fluorescence light it isgenerally preferred that the one or more lens(es) used for focusing oflight exposed onto an array of detection elements is substantiallytransparent to light in the wavelength regions of between 200 nm and1,000 nm, preferably where it is transparent in the wavelength region ofbetween 300 nm and 1,000 nm more preferably where it is transparent inthe wavelength region of between 350 nm and 850 nm. Preferably the oneor more lens(es) used for focusing are optically aberration corrected inthe range. Preferably the transparency of the one or more lens(es) issuch that attenuation of light is less than 3 OD in the region, morepreferably less than 1 OD.

While emitted light such as fluorescence light are typically weak inintensity, transmitted light, such as light used to determineattenuation is often of considerable intensity. In many embodiments itis therefore often preferred to attenuate the light through the use ofan optically dampening filter, preferably where the light emitted ontothe sample compartment is attenuated. Preferably the dampening filterused has properties that reduce the transmitted light intensity by morethan 1 OD, preferably reducing the intensity of transmitted light by asmuch as 3 OD.

The array of active detection elements in embodiments of the presentinvention is typically either a CCD or CMOS sensor.

In several preferred embodiments of the present invention, where inaddition to light attenuation image, two or more florescence lightintensity images are recorded, the fluorescent light is filtered usingwavelength limiting means, such as filter and/or interference filters itis preferred that these filters can be interchanged between recording ofimages. These filters could be mounted on a fixture that is moved, suchas by linear translation or by rotation.

In preferred implementation of the present invention the opticalmagnification of light exposed onto an array of active detection elementis less than 20:1, defined as the ratio of size of projection of anydimension of an object in the sample compartment to the size of theobject. Other equally preferred embodiments have optical magnificationof between 1:1 and 20:1, preferably between 1:1 and 1:10. In severalembodiments where large are of the sample compartment are imaged it ispreferred that the optical magnification is less than 4:1, such as inthe range from 1:1 to 4:1.

While several embodiments of the present invention include means withfixed optical magnification, several equally preferred embodimentsinclude means which allow recording of images at two or more opticalmagnifications, which for instance can be used to facilitate detectionof cells at lower magnification and subsequent detail analysis of cellsat higher magnification. Some preferred embodiments include means forvariable optical magnification, e.g. zoom.

As many of the preferred embodiments of the present invention includeexposing a number of light intensity images for the purpose of assessingproperty of a biological property it is preferred that any movement of asample or particle in a sample is kept under control, more preferablysuch movement should be kept at minimum. Therefore it is preferred thatthe volume of liquid sample is at stand still during the exposure, wherestand-still is defined as the situation where at least a part of theimage of a biological particle does not move any more than it iscontained substantially within the boundary of the same detectionelements during one exposure. Further when more than one exposer oflight onto the array of detection element, for the generation of two ormore images of light intensity information, it is preferred thatconditions of stand-still are maintained, where stand-still is definedas the situation where at least a part of the image of a biologicalparticle does not move any more than it is contained substantiallywithin the boundary of the same detection elements during time of twoexposures, preferably such that it is contained substantially within theboundary of the same detection elements during time of more than twoexposures, such as during three, four, five or even six exposures. Mostpreferably conditions of stand-still are maintained during the exposureof all images of light intensity information processed for theassessment of property of biological particle.

In preferred method of the present invention the volume of liquid sampleis at stand-still during the exposure, more preferably stand-still isbefore, during, after exposure, and therebetween. Stand-still conditionsare conditions where nothing moves but such conditions can be difficultto obtain in a liquid system such as when biological particles aresuspended in liquid, since there might be forces such as gravitational,or kinetical in play causing parts of the sample, including particles tomove, e.g. through sedimentation or oscillation, such forces acting onthe sample unintentionally, that is without active activation and/orcontrol. It is therefore often preferred to define stand-still whereconditions where no intentional force is applied to the sample, samplecompartment or detection means which can cause movements. Anotherpreferred definition is where stand-still is defined as the situationwhere at least a part of the image of a biological particle does notmove any more than it is contained substantially within the boundary ofthe same detection elements during time of an exposure with a lightsource, such as through the time of exposure with first or a secondlight source, preferably where the cause of movement is unintentional.

Another equally preferred definition of stand-still is where stand-stillis defined as the situation where the sample does not move relative tothe array of active detection elements such that the image of abiological particle in the sample does not move any more than it iscontained substantially within the boundary of the same detectionelements during the gathering of light intensity information, preferablyduring two or more exposures, preferably where the cause of movement isunintentional. Still another equally preferred definition of stand-stillis where stand-still is defined as the situation where the sample doesnot move relative to the array of active detection elements such thatthe image of a biological particle in the sample does not move any morethan it is contained substantially within the boundary of the samedetection elements during the gathering of light intensity information,preferably during two or more exposures, preferably where the cause ofmovement is unintentional.

One often preferred method to obtain stand-still conditions is to allowthe sample with suspended biological particles to stand for sufficienttime after movement of the sample and/or the sample compartment beforeinitiating measurement, in order for biological particles in suspensionto sediment and/or float to the inner lower and/or upper boundaries ofthe sample compartment, thus obtaining stand-still conditions ofbiological particles relative to the sample compartment. Preferably thesedimentation and/or flotation of biological particles is realised in arelatively short time, such as less than 240 seconds, preferably lessthan 100 seconds, such as in 45 seconds. More preferably, the settlingtime is longer than 10 seconds, preferably in the range between 10 and240 seconds, more preferably in the range between 30 and 120 seconds.Stand-still may be defined as the situation where the sample does notmove relative to the sample compartment. In a preferred embodiment ofthe present invention, the stand-still is defined as over a period oftime such as of least 10 seconds, such of at least 9 seconds, such of atleast 8 second, such of at least 7 seconds, such of at least 6 seconds,such of at least 5 seconds, such as at least 4 seconds, such as at least3 seconds, such as at least 2 second, or such as least 1 second.Preferably, this period may be before, during and/or after exposure.

The volume of sample analysed usually relates to the statistical qualityof the assessment of biological particles, since the size of the volumetypically correlates directly to the number of individual particles thatare analysed. For instance when the assessment of biological particlesconcerns the counting of individual particles the total number ofcounted particles determines the precision of the results. One parameterwhich has influence on the volume of sample analysed is the thickness ofthe sample compartment, defined by its wall parts, and therefore preferseveral embodiments of the present invention that the interior of thesample compartment has an average thickness of between 20 μm and 1,000μm. In these and other preferred implementations have average thicknessis between 20 μm and 100 μm. Ideally the thickness of the samplecompartment is uniform, but it has surprisingly been found that asubstantial deviation from uniform thickness does not compromise theresults of the assessment, as long as the average thickness of theportion of the sample compartment that is analysed is known. Further, inembodiments where the assessment of biological particles is performed ina substantially disposable sample compartment, such as samplecompartments intended for only a single analysis of a sample, it ispreferred that the average thickness of each sample compartment isknown, or preferably determined by means of the system.

In preferred implementations of the present invention the volume of theliquid sample from which electromagnetic radiation is exposed onto thearray of detection elements is in the range between 0.01 μL and 20 μL,such as between 0.05 μL and 5 μL. As the total volume of the samplebeing assessed, which is exposed onto the array of detection elementsdepends on several factors, including the sample thickness and area ofthe part of the sample compartment that is exposed onto the array ofdetection elements, it is possible to combine several features of thepresent invention in order to determine this volume but many of thepreferred embodiments result in volume that is analysed in a singleexposure that is between 0.05 μL and 1.0 μL. The total volume of theassessment can preferably be further increased by exposing additionalportion of the sample, either by replacing the portion of the sample inthe sample compartment with a different portion or by moving the samplecompartment and thus exposing a different part of the sample compartmentonto the array of sample compartment. Placement or replacement of thesample or a portion of the sample may be achieved by pumping the sampleor the portion of the sample into the sample compartment with pumpingmeans such as a pump, such as a plunger, and/or such by means of forcessuch as capillary forces and/or such as the gravitational force. Afterplacement or replacement of the sample or a portion of the sample, thesample or the portion of the sample is stationary inside the samplecompartment, preferably stationary due to the absence of any intentionalaction such as the application of a force to the sample or a portion ofthe sample, and that the sample compartment can be moved around so as toanalyse and/or assess the sample or the portion of the sample accordingto the present invention.

One generally preferred embodiment, which typically has a significantcontribution to the determination of the volume of sample exposed ontothe array of detection elements is the view area of the opticalarrangement exposing light intensity information, include embodimentswhere the view area are substantially fixed, or in equally preferredembodiments where the view area can be determined on the bases of theadjustment of the optical components. One preferred method for thedetermination of the volume analysed in a single exposure is to combineinformation concerning the thickness of the sample compartment withinformation about the active view area of the exposing system. Accordingto an embodiment of the present invention, the thickness of the samplecompartment is determined individually for the sample compartment inuse. Furthermore, the sample compartment may be intended for a singleanalysis of a sample, and in many preferred embodiment the samplecompartment can only be used for the analysis of a single sample.

Biological particles are diverse in type and properties, but it isgenerally preferred in several of the embodiments of the presentinvention that the size of the particles, the parameter or parameters ofwhich is/are to be assessed, are of a size between 0.1 μm and 100 μm.Such size of a particle is typically the average diameter of a particle,and in several equally preferred embodiments this average size of abiological particle is between 0.1 μm and 20 μm. In other preferredembodiments of the present invention, the size is between 5 μm and 15μm. In some embodiments of the present invention, the size is between 1μm and 15 μm, such as at least 15 μm, at least 14 μm, at least 13 μm, atleast 12 μm, at least 11 μm, at least 10_(i).tm, at least 9 μm, at least8 μm, at least 7 μm, at least 6 μm, at least 5 μm, at least 4 μm, atleast 3_(i).tm, at least 2 μm, or such as at least 1.

As diversity of biological particles is large as well as properties ofsuch particles, embodiments of the present invention can be used toassess a great number of quantity and/or quality parameter of biologicalsample and/or particles of a biological sample. Among several of suchpreferred parameters are; the number of the biological particles pervolume of a liquid sample, the diameter, area, circumference, asymmetry,circularity of the biological particles, determination of adhesionand/or degree of clumping of biological particles, preferably wheredegree of clumping allows the substantial determination of the number ofindividual cells in a clump of cells. Other equally preferred parametersare; the species of biological particles, the metabolic status ofbiological particles, intracellular property, such as number, size,shape of nucleolus.

Further one property of biological particle in a sample, often preferredwhere the assessment comprises the recording of two or more images ofspatial light intensity information, where one image representsattenuation of light, is the substantial location of a biologicalparticle in the spatial light intensity image. This location of aparticle is preferably used to correlate other light intensityinformation to the particle, such as fluorescence. This is for instanceoften preferred when among plurality of individual particles it can beexpected that some particles reflect such other light intensityinformation while other particles substantially do not reflect thislight intensity information. The substantial absence of such lightintensity information can make it difficult to determine the presence ofsuch particle based solely on the light intensity information, as itsometimes shows no information which can be differentiated from thebackground. In these instances it is often preferred that the locationof a particle can be derived on the bases of other image of spatialimage intensities.

Preferred embodiments of the present invention include methods for theassessment of properties of biological samples and/or biologicalparticles, where an image of spatial light attenuation information isrecorded. Many of these embodiments preferably further include the stepsof recording additional images of spatial light intensity information,included in the processing of image information, where the additionalspatial light intensity information is information about fluorescence.In several embodiments this additional fluorescence image information isgenerated by excitation light from the light source passed through thewall parts of the sample compartment and used to generate attenuationimage information, where an emission filter is employed thus producingfluorescence. In other equally preferred embodiments such additionalfluorescence information image is generated by excitation light from anadditional light source. As more information is generally recorded byrecording more than one or two images of spatial light intensityinformation, it is generally preferred that in the addition to an imageof attenuation information, two additional fluorescence informationimages are included in the processing of images, and typically it ismore preferably to use three, four or five additional fluorescenceinformation images. According to one embodiment of the presentinvention, an image of spatial light intensity information is recorded,where the spatial light intensity information is information aboutfluorescence, caused by excitation light from the second light sourcepassed through the wall parts of the sample compartment. In otherpreferred embodiments the second spatial fluorescence light intensityinformation is caused by excitation light from a third light source. Inanother embodiment of the present invention, a third image of thirdspatial light intensity information is recorded, where the third spatiallight intensity information is information about fluorescence, caused byexcitation light from the second or third light source passed throughthe wall parts of the sample compartment, preferably caused byexcitation light from a fourth or subsequent light source. In a thirdembodiment of the present invention, a fourth image of fourth spatiallight intensity information is recorded, where the fourth spatial lightintensity information is information about fluorescence.

In embodiments of the present invention, where parameter of a biologicalsample and/or biological particle include multiple images are includedin the processing it is usually preferred to use two or more lightsources in order to obtain the image information needed. The number andnature of the light sources, which are usually two, three, four or fiveindividual light sources, reflects properties such as wavelength andintensity of light, attenuation and scatter, and in the case offluorescence background suppression.

In several highly preferred embodiments of such multi-image processingit is preferred that spatial information about location of biologicalparticles is an integrated feature of the processing of images ofspatial light intensity information.

In embodiments including processing of multi-image information,parameters of biological particles that can be assessed can preferablybe one or several of the following; assessment of species biologicalparticle, condition of biological particle, preferably wherein conditionof biological particle is metabolic condition such as cell cycle,viability, vitality, apoptosis, motility. In a preferred embodiment ofthe present invention, the location of biological particles in the firstspatial light intensity image is used to determine presence of lightintensity information in another recorded image of light intensityinformation associated to biological particles, preferably where theother light intensity information is fluorescence. In another preferredembodiment of the present invention, the location of biologicalparticles is determined by combining information in a first and a secondimage of spatial light intensity information, where the images arerecorded using illumination from the first light source and applyingsubstantially different modulation means for the images, preferablywhere the images are dark-field and bright-field images. In yet anotherpreferred embodiment of the present invention, the location ofbiological particles is determined by combining information in three ormore images of spatial light intensity information, where the images arerecorded using illumination from the first light source and applyingsubstantially different modulation means for the images. Thedetermination of intensity of a second or additional light intensityinformation associated to biological particles may be used for theassessment of species and/or condition of biological particle.Alternatively, the determination of intensity of a second or additionallight intensity information associated to biological particles may beused for the assessment of species and/or condition of biologicalparticle comprising a bio marker.

In many preferred embodiments of the present invention a light sourceused is a tuneable solid-state light source. The presence of a tuneablelight source can significantly simplify the design of system accordingto the present invention, and would also allow for greater flexibility,such as when the tuneable solid-state light source is used forexcitation of fluorescence light. Preferably such tuneable solid-statelight source is either a light emitting diode (LED) or a laser diode.

EXAMPLE 1 Image Cytometer

FIG. 1 illustrates possible configuration of an Image Cytometerincluding several preferred embodiments of the present invention. Theillustration outlines 4 main groups of components, the sample holder(100), illumination means (110), imaging means (120) and detection means(130). Finally it illustrates the main optical axis of the ImageCytometer (140), along which majority of the optical components arearranged.

The sample holder can be moved along the optical axis relative to theimaging means, in order to assure that the sample is in focus alignmentwith the imaging means. The sample compartment (101) is placed on thesample stage (102) in the optical path. The sample compartment istypically attached to the sample stage but it can be released there fromsuch that it can be removed from the Image Cytometer and replaced againthrough either manual or automated process. The sample stage can move intwo directions perpendicular to the optical axis. This allows differentparts of the sample inside the sample compartment to be assessed.

The illumination means can move along the optical axis in order tomaintain the desired illumination of the sample compartment. Theillumination means contains usually 2 or more light sources (3 shown).The illustration shows a light source located on the optical axis (111),such that illumination of the sample in the direction towards thedetector, which is often only preferred when the purpose is to generatean image of passive light attenuation and/or scattering properties, suchas Bright-field or Dark-field images. In addition the illustration showstwo fluorescent light sources (112), which illuminate the samplecompartment at an inclination relative to the optical axis, which hasbeen found to improve conditions under which signal from particles isidentified as being different from signal from the background. Itfurther illustrates a light source emitting a single wavelength (112 a)as well as a light source emitting two wavelengths (112 b), by placingtwo Light Emitting Diodes in a single arrangement.

The imaging means comprise collection objective (121) in an arrangementwhere it is possible to interchange two or more collection objectives(121 a and 121 b) with substantially different properties, such asnominal magnification. The two or more collection objectives areinterchanged either by linear or circular movement. Light modulationmeans are preferably two ore more and contain a number of opticallyactive components labelled Y_(i) (122) and X_(i) (123) respectively,such as filters, apertures, obstructions or phase contrast elements. Thelight modulation means can be moved perpendicular to the optical axissuch that each of the optical components can be placed in the beam oflight emitted from the collection objective, the movement can either belinear or circular. Each light modulation means preferably has aposition without an optical component, such that if all light modulationmeans are arranged such that this empty position is located in the lightbeam then no modulation takes place. The imaging means contain focusingmeans (124) which focus the light from the collection objective onto thedetector.

The detection means can be moved along the optical axis in order recorda focused image of light intensity information. The information isgathered using an array of active detection elements (131), a lightsensitive camera.

The operation of the Image Cytometer and collection of data iscontrolled by computer means (not shown). The computer means preferablyis equipped with image processing means which can be used for automaticidentification and assessment of biological particles.

EXAMPLE 2 Properties of Low Wavelength Microscopy

Contrast in Bright Field microscopy according to the present inventionwas investigated by measuring a sample of Jurkat cells (human leukemiacell line, subclone A3, ATCC CRL-2570). The measurements were performedusing four light sources of different wavelength. Three of the lightsources were single colour narrow-waveband Light Emitting Diodes (LEDs)and the fourth light source was a broad- waveband white LED. All lightsources were in optical arrangement where emitted light was collimatedwhen passing the sample.

The output from the narrow- waveband LEDs was used without modificationbut the output from the white LED was used with modification, as well asbeing modified using narrow-band filters. The wide- waveband light fromthe white LED represents typical conditions of visible microscopy. Theprinciple wavelengths of the narrow-waveband light used in themeasurement are listed in the following Table 2-1.

TABLE 2-1 List of Light Sources Light Source Principal wavelength LED365 nm 365 nm LED 400 nm 400 nm LED 453 nm 453 nm White + Filter 555 nmWhite + Filter 720 nm

The sample containing the Jurkat cells in suspension was loaded into asample compartment of about 100 μm thickness. The sample compartment wasplaced in the optical system and the bright field information wasfocused using a 2× linear magnification onto an array of activedetection elements. The focus and light intensity of each of the imageswas adjusted to produce comparable results.

Information in the images was analysed by determining the TotalIntensity Contrast of individual cells, as the ratio of integratedintensity of a cell to the intensity of the background, which is ameasure of the relative attenuation of light. The results of thecontrast determination are presented in the graph in FIG. 2A, whichshows the observed contrast as a function of waveband of the lightsource. In the graph a solid line is drawn in the wavelength range from400 nm to 750 nm which represents the observed Total Intensity Contrastwhen using the broad-waveband light of the white LED.

FIGS. 2B through 2G show examples of the recorded images. FIG. 2B isimage recorded using light of 365 nm, 2C using light of 400 nm, 2D usingwhite light and FIG. 2E using light of 710 nm. The images show that thecontrast in the images has profound influence on the imagerepresentation of the biological particles for the purpose of accurateidentification of the presences and spatial position of the cells.

FIGS. 2F and 2G show sections of the collected images in higherresolution. In FIG. 2F it is the image using 365 nm light and in FIG. 2Git is the image using 710 nm light. The images show that using light atwavelength below 400 nm results in an image that shows more details thanimage collected using long-wavelength light, although images arecollected under similar conditions. Image of 365 nm light shows fairamount of details about shape, size and relative position than does thelong-wavelength image.

EXAMPLE 3 Enhancement of Signal to Background

When performing fluorescence analysis of a biological sample it isimportant to manage excitation and emission light in order to obtainadequate contrast in the image. There are basically two approaches toimproving contrast, firstly to use an excitation filter to reduce lightof wavelength longer than what is used for the excitation offluorescence and secondly to use an emission filter to reduce light ofwavelength shorter than the fluorescent light from reaching the array ofactive detection elements.

In order to perform high-sensitivity fluorescence analysis, where signalcontrast is an important aspect, it is necessary to consider all aspectsof the optical system which affect the collected intensity information.This includes aspects such as excitation intensity, auto-fluorescence ofany component of the system and attenuation of optical filters. In thetask of improving contrast in collecting fluorescence spatial intensityinformation of a biological sample it is of importance to firstlyoptimise the intensity of fluorescence signal and secondly to minimisebackground signal. The intensity of fluorescence signal is mainlydetermined by the intensity of excitation light. The intensity ofbackground signal is dependent on several aspects such as exposure ofexcitation light onto the array of active detection elements andintensity of auto-fluorescence of optical components.

Ideally exposure of excitation light onto the array of active detectionelements can be eliminated by the use of an emission filter withinfinite attenuation or blocking. Such ideal filters are difficult torealise and filters generally attenuate light to a fraction at a givenwavelength, such as 10⁻⁶ to 10⁻⁷ (attenuation of between six and sevenorder of magnitude), and therefore in addition to high quality filtersit is necessary to consider other aspects which affect exposure ofexcitation light. The orientation of the excitation light sourcerelative to the field of view of the array of active detection elementscan have great influence on the exposure of excitation light onto thedetection elements, where general orientation directly along the axis offield of view of the detection elements will normally give rise tohighest intensity of exposed excitation light. General orientation ofexcitation light off the field of view axis will reduce the intensity ofexposed excitation light onto the detection elements.

In a preferred embodiment of the present invention the excitation lightis directed towards the sample in the sample compartment at an angle tothe axis between the sample compartment and the array of activedetection elements. Such an embodiment is shown in FIG. 3A where thegeneral axis of the excitation light is at about 40 degrees to the axisbetween the sample compartment and the array of active detectionelements. In FIG. 3A shows a collection objective (301) which collectslight from the sample compartment, defined by a transparent wall part(303) defining the bottom of the sample compartment and anothertransparent wall part (304) defining the top of the sample compartment.The objective transmits the light from the sample compartment through anemission filter (302) and images it onto the array of active detectionelements (not shown).

The excitation light is produced by a Light Emitting Diode (305), thelight from which is collected by the first lens in the excitation lightmodule (306), which consists of one or more lenses (two shown) and oneor more light modulation elements (two shown), such as excitation filterand light dispersive element. The light from the excitation light moduleis focused onto the sample compartment as indicated by the boundary ofthe light beam of the excitation light (307). Under conditions such asthose given in the figure, it is possible that a part of the excitationlight can enter the field of view of the collection objective, either byscattering (not shown) or by direct illumination which can occur if apart of the excitation light beam enters the opening of the collectionobjective (307 a) at angles below the acceptance angle. A great fractionof this light is removed by the emission filter before it reaches thedetection elements (309) while fluorescent light is allowed to reach(310). Although the emission filter has great attenuation it is limited,for instance to a factor of 10⁻⁷, which means that this fraction of thelight, although small, can reach the detection elements. Further theelements of the collection objective can produce fluorescence(auto-fluorescence) which will pass the emission filter and produce abackground image on the detection elements.

FIG. 3B gives an illustration of the light intensity at the entry of thecollection objective corresponding to the fraction of the beam ofexcitation light (307 a). From the arrangement of the components of theexcitation module it would be possible to reduce this light by limitingthe aperture of the excitation light module but this would greatlyreduce the total amount of excitation light exposed onto the samplecompartment and thus similarly reduce the fluorescence intensity. Apreferred embodiment of the present invention is illustrated in FIG. 3C,where an obstruction(s) (311) is/are placed in the excitation lightmodule, thus changing the light beam of the excitation light (312) whichlargely removes the rays of light which would enter the field of view ofthe collection objective.

FIG. 3D shows the intensity profile of the modified light beam ofexcitation light, where a fraction is removed by the obstruction. FIG.3E show the resulting light intensity entering the collection objective,and comparison to FIG. 3B it shows that the extension of light has beengreatly removed but further in this example it is important to note thatthe scaling of the two images is such that FIG. 3E has been amplified bya factor of approximately ×200, illustrating that the intensity of theexcitation light has been reduced effectively.

In a preferred embodiment of the present invention the excitation lightis directed towards the sample in the sample compartment at an angle tothe axis between the sample compartment and the array of activedetection elements. Such an embodiment is shown in FIG. 3F which showsthe same configuration of the excitation light source and opticalelements as in FIG. 3A. The configuration in FIG. 3F further shows acover (315), e.g. in the form of an aperture, for reducing the amount ofexcitation light reaching the objective. The embodiment in FIG. 3Ffurther shows a light source (313) located on the optical axis extendingfrom the array of active detection elements to the sample compartment.This configuration is similar to the one shown in FIG. 5A. The lightfrom the light source in FIG. 3F was passed through optical means suchthat it formed a beam of light (314) that was substantially parallel tothe optical axis of the collection objective such that the beam of lightis collimated.

The following Table 3-1 shows the effect of the size of the obstructionon the intensity of the excitation illumination and the intensity ofexcitation light entering the collection objective. The table shows therelative amount of excitation light exposed onto the sample compartmentas well as the relative amount of the light beam of excitation lightthat enters the collection objective. The obstruction is formed byplacing a linear screen into the cylindrical beam of light inside theexcitation light module and the reported values are the insertion of theobstruction relative to the diameter of the light beam.

TABLE 3-1 Excitation Light and Light in Collection Objective ObstructionExcitation Light Light in Objective  0% 100%   11%  6%  98%    8% 13% 93%    3% 19%  85%  0.8% 22%  81%  0.18% 25%  77% 0.005%

The effect in the analysis of weakly fluorescent particles, for instanceparticles containing relatively few fluorochromes, is demonstrated inthe following using typical conditions. In an embodiment of the presentinvention let us assume the use of a collection objective with NumericalAperture (NA) of 0.20. Further let us assume that fluorescenceconversion efficiency of the particles is 2×10⁻⁶ and similarly that thefluorescence conversion efficiency of the sample compartment is 2×10⁻⁷,this fluorescence originating from impurities in the transparent wallpart and/or other materials. The emission filter attenuates majority ofthe excitation light entering the collection objective but upon enteringthe collection objective the light can give rise to substantialbackground fluorescence, caused by impurities in optical components aswell as other materials in the collection objective, and substantialpart of this fluorescence will pass through the emission filter.Therefore there is a net light intensity transmitted through theemission filter onto the detection elements, caused by excitation lightentering the collection objective, and we can assume that the netintensity of scattered, exposed and fluorescent light passing theemission filter amounts to 5×10⁻⁷.

These conditions describe a system where it is very difficult to assessfluorescence intensity from a biological particle, since the totalintensity of the background is approximately 3 times that of thefluorescence intensity observed from the particle. By inserting anobstruction the beam of excitation light in the excitation light moduleit is possible to reduce the amount of light entering the collectionobjective and thus suppress the background signal significantly. InTable 3-1 we have shown that at the same time the total amount ofexcitation light entering the sample compartment is also reduced to amuch less extent. The effect of this illustrated in the following Table3-2, which shows normalised fluorescence and background signals.

TABLE 3-2 Normalised Fluorescence and Background Signals Obstruc- Fluor-Sample Light in Back- Signal/ tion escence Background Objective groundBackground  0% 1.00 0.10 2.67 2.77 0.36 (1)  6% 0.98 0.10 1.98 2.08 0.47(1.3) 13% 0.93 0.09 0.74 0.83 1.1 (3.1) 19% 0.85 0.09 0.16 0.24 3.5 (10)22% 0.81 0.08 0.04 0.12 6.8 (19) 25% 0.77 0.08 0.00 0.08 9.6 (27)

Table 3-2 shows that the total background signal reduces significantlymore rapidly than the fluorescence intensity, which is illustrated inthe Signal/Background (S/B) values (in parenthesis is the relativechange in the ratio of fluorescence signal to background signal). Theresults of Table 3-2 suggest that under these conditions the S/B ratioincreases almost 30 fold by placing an obstruction in the beam ofexcitation light than covers about 25% of the diameter of the lightbeam. Under these conditions the intensity of fluorescent light hasdeclined by only 23%.

EXAMPLE 4 Detection of Particle Standard

A system according to the present invention was used to identify andquantify calibration beads, which typically are used to calibrate flowcytometry instruments. The beads were Rainbow Calibration Particles (P/NRCP-30-5A), Spherotech USA a set of beads 3 μm in diameter comprising 8groups producing different intensities of fluorescence.

The image cytometer of the present invention was set to illuminate thesample with excitation light in a narrow band around 475 nm and todetect fluorescence emission in a waveband at around 536 nm. Light fromthe sample compartment was collected using 4× collection objective withNA 0.20 and focused onto the array of active detection elements. Thebeads were handled according to instructions provided by the supplierand placed in a sample compartment of approximately 100 μm thickness.

Series of images of spatial light intensity information were collectedby an array of active detection elements, firstly an exposure of spatialbright field light intensity information was collected for the purposeof acquiring information concerning the position of the particles andsecondly an exposure of spatial fluorescence light intensity wascollected using integration time of 300 ms. A total of 35 pairs ofbright field and fluorescence images was collected from different partsof the sample by moving the sample compartment, resulting in a total of70 light intensity images.

Each bright field image was used to determine the position of a particleand this information was used to interrogate the fluorescence image atthat location, integrating the total intensity of fluorescent light fromeach particle. A total of 12,750 particles were analysed and the resultsare presented in FIG. 4, which shows a histogram of observed fluorescentintensities. The figure shows clear distinction of 8 different intensitygroups, in compliance with the specifications given by the suppliers ofthe calibration beads. This demonstrates that sensitivity of an imagecytometer according to the present invention is similar to a typicalflow cytometer of the day.

EXAMPLE 5 Bright-Field/Dark-Field Configuration

A sample of adherent WeHi-S cells (murine fibrosarcoma cell line) wasplaced in an image cytometer of the present invention depictedschematically in FIG. 5A and illuminated with light source (503)emitting light in a narrow waveband around 365 nm. The light source waslocated on the optical axis extending from the array of active detectionelements (509) to the sample compartment defined by two transparent wallparts (502 and 503) exposing light towards the detection elements.

The exposed light was passed through optical means (507) such that itformed a beam of light (504) that was substantially parallel to theoptical axis of the collection objective (501). The properties of thecollection objective used is such that the parallel light emitted fromthe light source is substantially focused onto a plane (506) locatedalong the optical axis between the objective and the array of activedetection elements such that a substantial part of that light isdirected away by the collection element (508) that focuses light ontothe detection elements. Using this arrangement a portion of the lightemitted from the light and passed through the collection objectiveenters the detection elements forming uniform background intensity,unless if that light is deflected by a particle in the sample then lightfrom that location of the sample attenuates the light intensity thusforming a spatial bright field image of the particle.

The light that is deflected by the particle is affected by the elementsof the particle. The light such deflected changes direction and it isemitted in several directions, the intensity of the light in differentdirections being determined by the properties of the particle. Some ofthe deflected light enters the collection objective and forms a bundleof light (505) of different directions originating from the particle.The deflected light entering the collection objective forms asubstantially parallel beam of light when it reaches the collectionelement, which will be focused onto the collection elements. Thisdeflected light forms a spatial image of light intensity informationthat is mixed with the bright field image of the sample, an example ofwhich is given in FIG. 5D.

FIG. 5B shows an obstruction (510) that is placed in the focal plane ofthe substantially parallel light emitted from the light source, locatedbetween the collection objective and the detection elements. Thisobstruction is an aperture formed by a hole in a disk, the dimension ofthe hole being such that it substantially only allows light from thelight source to reach the detection elements eliminating light dispersedby the particles. The resulting image, an example of which is given inFIG. 5E, is a spatial image of bright field light information, wherecontrast is substantially improved compared to an arrangement withoutsaid aperture obstruction discussed previously. An additional propertyof this arrangement is that such bright field image is considerably lesssensitive to focusing of the collection objective, since focusing ofthis bright field image is largely dependent on the degree ofcollimation of the light passing through the sample compartment and thedimension of the aperture of the obstruction, thus facilitating imageswith substantially large focus depth.

FIG. 5C shows an obstruction (511) that is placed in the focal plane ofthe substantially parallel light emitted from the light source, locatedbetween the collection objective and the detection elements. Thisobstruction has dimensions and is located such that it substantiallyonly allows light dispersed by the particles to reach the detectionelements eliminating light emitted directly from the light source. Theresulting image, an example of which is given in FIG. 5F, is a spatialimage of dark field light information of the particles in the sample.

FIG. 5G illustrates rays of light interacting with a spherical objectwhich properties, with regard to refractive index, are similar to thoseof a biological particle. It shows that when collimated light (520)illuminates the particle (521) that the rays of light are refraction dueto difference in refractive index, the degree of refraction beingdetermined by the optical properties of the particle. The light passingaround the particle (522) enters the collection objective (not shown)still collimated. Some of the light is dispersed at large angles (523),such that they will illuminate the plane of the entrance of thecollection objective outside the entrance, thus not reaching the arrayof detection elements (not shows). Other rays are refracted at smallangles (524), such that they will enter the collection objective andthus being imaged on the detection elements.

Considering the collimated rays of light which pass around the particle,these will form an image on the detection elements with considerablelight intensity outside the image of the particle and small or no lightintensity inside the particle, thus forming a shadow of the particle.The rays of light interacting with the particle are refracted to adifferent degree, some of which have zero angle of refraction, sincethey cross the boundary of the particle at perpendicular, while othershave varying angle of dispersion. Considering dispersed rays of lightthese will form an image of the particle, to some extend similar to animage which would be observed if the particle were luminous, but with adifferent point of dark-field focus (525) not coinciding with theposition of the particle, the point of focus being determined by thesize and optical properties of the particle.

FIG. 5H through FIG. 5J show images of CHO cells in suspension recordedusing different illumination condition. FIG. 5H is a bright-field imageof the cells, while FIG. 5I is a bright-field image where refractedlight has been blocked by placing an aperture at the focus plane ofparallel light (see FIG. 5B) and FIG. 5J is a dark-field image wherecollimated light has been blocked (see FIG. 5C), all images are focusedindividually. Finally FIG. 5K is a fluorescence image of the particlesshowing specific staining. The images in FIG. 5H, 5I and 5J are used incombination to determine location and outlines of individual cells,information that is used to count the number of cells and to estimatefluorescence intensity of individual cells, which is used to classifycell property.

By arrangement where the obstruction(s) in the focal plane of thecollection objective are interchangeable it is possible to realise animage cytometer system that has the flexibility of recording highcontrast bright field or dark field, or the combination of the two,simply by placing or removing a suitable obstruction.

1. An imaging apparatus, comprising: a first light source configured for emitting light into a sample region; a collimator for forming collimated light from the first light source and directing the collimated light along an optical axis of the imaging apparatus and into the sample region; a second light source comprising a first excitation light source configured for emitting excitation light into the sample region; an image forming element for forming at least one image of at least part of the sample region on an array of detection elements; wherein the sample region is located between the collimator and the array of detection elements; wherein the imaging apparatus is configured to be interchanged between a bright field mode, a dark field mode and a fluorescence mode.
 2. The imaging apparatus according to claim 1, wherein the first light source provides the light for the bright field mode and the dark field mode.
 3. The imaging apparatus according to claim 1, further comprising a modulator located between the sample region and the array of detection elements, the modulator operable to interchange between forming the bright-field image and the dark-field image.
 4. The imaging apparatus according to claim 3, wherein the modulator comprises an aperture configured for attenuating light during the formation of the bright-field image, wherein the light is passing the sample region as collimated light.
 5. The imaging apparatus according to claim 3, wherein the modulator comprises an obstruction configured for attenuating light leaving the sample region as collimated light during the formation of the dark-field image.
 6. The imaging apparatus according to claim 5, wherein the obstruction comprises an at least partly opaque centre surrounded by an at least partly transparent area.
 7. The imaging apparatus according to claim 3, wherein the image forming element is located between the sample region and the array of detection elements, and the modulator is located at or close to the focal plane of the image forming element.
 8. The imaging apparatus according to claim 6, wherein the at least partly opaque centre is located in or close to a focal point of the image forming element.
 9. The imaging apparatus according to claim 3, wherein the modulator comprises a phase contrast microscopy modulator.
 10. The imaging apparatus according claim 1, wherein the excitation light is at an incidence angle between 10 and 80 degrees, or of 90 degrees, or between 110 and 180 degrees relative to the optical axis so as to provide the fluorescence image.
 11. The imaging apparatus according to claim 1, wherein the first and/or second light source(s) is/are configured for emitting light in duration between 0.0001 and 0.1000 second.
 12. The imaging apparatus according to claim 1, wherein the first and/or second light source(s) is/are configured for emitting light in duration for more than a 1 second.
 13. The imaging apparatus according to claim 1, wherein the light source(s) is/are optically connected to an optical means configured for providing light with a substantially uniform intensity across the sample region and/or across a region imaged by the array of detection elements.
 14. The imaging apparatus according to claim 13, wherein the optical means comprises an array of micro lenses.
 15. The imaging apparatus according claim 13, wherein the optical means comprise two arrays of cylindrical micro lenses, wherein the lenses in one of the arrays are orientated with a major axis being perpendicular to a major axis of the lenses of the other array.
 16. The imaging apparatus according to claim 1, wherein the image forming element is configured for transmitting light in the wavelength region of between 200 nm and 1000 nm.
 17. The imaging apparatus according to claim 1, wherein the sample region is adapted to hold a liquid sample with biological particles.
 18. The imaging apparatus according to claim 1, wherein the image forming element is an objective and/or one or more lenses and/or one or more curved mirrors.
 19. The imaging apparatus according to claim 1, wherein the image forming element is configured for providing a linear enlargement of the sample.
 20. The imaging apparatus according to claim 19, wherein the linear enlargement is smaller than 20:1.
 21. The imaging apparatus according to claim 1, wherein the first light source and/or the second light source is/are a light emitting diode (LED) and/or a diode laser and/or laser.
 22. A method for the assessment of at least one quantity parameter and/or one quality parameter of a biological sample, comprising: applying a volume of the biological sample to a sample compartment having parallel wall parts defining an exposing area, the wall parts allowing light from an imaging apparatus to pass through the wall parts of the sample compartment, wherein the imaging apparatus comprises a first light source configured for emitting light into the sample compartment and a second light source comprising a first excitation light source configured for emitting excitation light into the sample compartment; illuminating the sample compartment with collimated light from the first light source, and exposing, onto a 2-dimensional array of active detection elements, light having passed through the sample compartment, thus recording a bright-field image of spatial light intensity information; illuminating the sample compartment with collimated light from the first light source, and exposing, onto a 2-dimensional array of active detection elements, light having interacted with the biological sample, thus recording a dark-field image of spatial light intensity information; illuminating the sample compartment with excitation light from the first excitation light source, and exposing, onto the 2-dimensional array of active detection elements, fluorescent light having passed through the sample compartment, thus recording a fluorescent image of spatial light intensity information; processing all three images in such a manner that light intensity information from individual biological objects are identified as distinct from light intensity information from the background; and correlating the results of the processing to the at least one quantity parameter and/or quality parameter of biological particles in the biological sample.
 23. The method according to claim 22, wherein two or more images of spatial light intensity information are recorded, each of the images being recorded with two or more substantially different modulators.
 24. The method according to claim 22, wherein the parameter to be assessed is location of biological particles in the spatial light intensity image.
 25. The method according to claim 24, wherein the location of biological particles in the first spatial light intensity image is used to determine presence of light intensity information in another recorded image of light intensity information associated to biological particles, where the other light intensity information is fluorescence.
 26. The method according to claim 24, wherein the location of biological particles is determined by combining information in a first and a second image of spatial light intensity information, where the images are recorded using illumination from the first light source and applying substantially different modulators for the images.
 27. The method according to claim 26, wherein the images are dark-field and bright-field images. 